The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

Summary ACOD1 (also known as IRG1) has emerged as a regulator of immunometabolism that operates by producing metabolite itaconate. Here, we report a key role of STING1 (also known as STING and TMEM173) in mediating ACOD1 expression in myeloid cells in response to toll-like receptor (TLR) signaling. The activation of STING1 through exogenous cyclic dinucleotides (e.g., 3′3′-cGAMP) or endogenous gain-of-function mutation (e.g., V155M) enhances lipopolysaccharide-induced ACOD1 expression and itaconate production in macrophages and monocytes, whereas the deletion of STING1 blocks this process. The adaptor protein MYD88, instead of DNA sensor cyclic GMP-AMP synthase (CGAS), favors STING1-dependent ACOD1 expression. Mechanistically, MYD88 directly blocks autophagic degradation of STING1 and causes subsequent IRF3/JUN-mediated ACOD1 gene transcription. Consequently, the conditional deletion of STING1 in myeloid cells fails to produce ACOD1 and itaconate, thereby protecting mice against endotoxemia and polymicrobial sepsis. Our results, therefore, establish a direct link between TLR4 signaling and ACOD1 expression through the STING1-MYD88 complex during septic shock.

The MYD88-STING1 protein complex is required for lipopolysaccharide (LPS)-induced ACOD1 expression The MYD88-STING1 protein complex prevents autophagic degradation of STING1 The IRF3-JUN transcription factor complex favors ACOD1 upregulation STING1-mediated itaconate production promotes experimental sepsis INTRODUCTION Sepsis is one of the oldest and most elusive syndromes in medicine that is defined by a dysregulated host response to pathogen infection (Singer et al., 2016). Monocytes and macrophages are the main sources of the production of immune mediators in septic shock, which can be activated by pathogen-associated molecular patterns (PAMPs) using a set of receptors called pattern recognition receptors (Wiersinga et al., 2014). Several metabolic processes in immune cells, including aerobic glycolysis, the tricarboxylic acid cycle, fatty acid metabolism, and itaconate metabolism, promote the activation or quiescence of the inflammatory response (Jung et al., 2019;O'Neill et al., 2016;Wu et al., 2022). Understanding the process, modulation, and function of immunometabolism is critical for the development of therapies for sepsis (Koutroulis et al., 2019).
In this study, we provide the first evidence that STING1 mediates LPS-induced ACOD1 expression by binding to adaptor protein myeloid differentiation marker 88 (MYD88), rather than through a CGAS-dependent signaling pathway. The deletion of STING1 in macrophages and monocytes limits LPS-induced ACOD1 expression as well as itaconate production, thereby preventing septic death in mice. These findings establish a framework for understanding the interaction of STING1 and TLR signals in the control of immunometabolism.
Next, we examined whether the constitutive activation of STING1 enhances LPS-induced ACOD1 upregulation. We focused on V155M, which is a gain-of-function mutation that leads to the constitutive activation of STING1 and subsequent immune-mediated inflammatory disease in humans (Jeremiah et al., 2014;Liu et al., 2014). Compared to wild-type cells, V155M-THP1 cells had an increased basic expression of interferon-alpha 1 (IFNA1, best known as IFNa) ( Figure 1G) and NF-kB target gene interleukin 6 (IL6) ( Figure 1H), rather than the basic mRNA expression of ACOD1 ( Figure 1I). However, V155M-THP1 cells became more sensitive to LPS-induced ACOD1 expression ( Figure 1I) and itaconate production ( Figure 1J). Importantly, LPS-induced ACOD1 protein expression and itaconate production were blocked in STING1 À/À THP1 cells and this phenotype was rescued by the re-expression of STING1 ( Figures 1K and 1L). Collectively, these findings strongly support the conclusion that STING1 plays a key role in regulating ACOD1 expression in LPS-activated monocytes and macrophages.
(G-J) WT and V155M-THP1 cells were treated with indicated LPS for 6 h, and then IFNA1 mRNA, IL6 mRNA, ACOD1 mRNA, and intracellular itaconate concentration were assayed (Data are presented as mean G SD; n = 3 biologically independent samples; two-way ANOVA with Tukey's multiple comparisons test).
(K) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) for 6 h. (L) In parallel, intracellular itaconate concentration was assayed (Data are presented as mean G SD; n = 3 biologically independent samples; two-way ANOVA with Tukey's multiple comparisons test).
(F) Western blot analysis of protein expression in indicated V155M-THP1 cells following treatment with LPS (500 ng/mL) for 6 h.
(G and H) Analysis of ACOD1 mRNA and intracellular itaconate concentration in indicated V155M-THP1 cells following treatment with LPS (50-5000 ng/mL) for 6 h (Data are presented as mean G SD; n = 3 biologically independent samples; two-way ANOVA with Tukey's multiple comparisons test).
The MYD88-STING1 protein complex prevents the autophagic degradation of STING1 As both MYD88 and STING1 are adaptor proteins in innate immunity, we hypothesized that STING1 and MYD88 may form a protein complex to regulate signal transduction. Indeed, immunoprecipitation analysis revealed that the STING1-MYD88 protein complex was present in THP1 cells, and this complex was mildly increased by stimulation with LPS/3 0 3 0 -cGAMP ( Figure 3A). Image analysis confirmed the colocalization between STING1 and MYD88 in RAW264.7 cells ( Figure 3B). However, the deletion of MYD88 increased LPS/ 3 0 3 0 -cGAMP-induced STING1 protein degradation in MYD88 À/À cells, whereas the depletion of STING1 had no effects on the level of MYD88 protein in STING1 À/À cells ( Figures 3C and 3D). Thus, the STING1-MYD88 complex prevents STING1 protein degradation in activated THP1 cells.

STING1-mediated itaconate production promotes experimental sepsis
To determine the significance of STING1-mediated ACOD1 expression in vivo, we used two mouse models, including one for endotoxemia as well as polymicrobial sepsis induced by cecum ligation and puncture (CLP). Consistent with our previous studies , the conditional deletion of STING1 in myeloid cells (termed Sting1 MyeÀ/À mice) prevented the animal death caused by endotoxemia ( Figure 6A) or CLP ( Figure 7A), and the production of circulating damage-associated molecular patterns  (0.1 ng/mL), imiquimod (5 mg/mL), ssRNA40 (5 mg/mL), or ODN2006 (10 mg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean G SD; n = 3 biologically independent samples; two-way ANOVA with Tukey's multiple comparisons test). (B) Wild-type THP1 cells were stimulated with indicated TLR ligands for 3 h and the mRNA level of TNF was assessed (Data are presented as mean G SD; n = 3 biologically independent samples).
(C) Wild-type THP1 cells were stimulated with indicated TLR ligands in the absence or presence of 3 0 3 0 -cGAMP (10 mg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean G SD; n = 3 biologically independent samples; two-way ANOVA with Tukey's multiple comparisons test).
(D) Wild-type and V115M THP1 cells were stimulated with indicated TLR ligands for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean G SD; n = 3 biologically independent samples; two-way ANOVA with Tukey's multiple comparisons test). The TLR ligand concentration used in panels B-D is the same as for panel A.

S t i n g 1 fl o x /f lo x S t i n g 1 fl o x /f lo x + 4 O
I G Acod1 mRNA (AU)

S t i n g 1 f lo x /f lo x S t i n g 1 f lo x /f lo x + 4 O
I G Acod1 mRNA (AU) iScience Article We next evaluated the impact of itaconate on an antiseptic phenotype of Sting1 MyeÀ/À mice. We administered 4-octyl itaconate (4OI), the cellular permeable derivate of itaconate, to Sting1 flox/flox (control group) and Sting1 MyeÀ/À mice. Unlike previous studies that showed that 50 mg/kg of 4OI can protect against endotoxemia (Mills et al., 2018), we did not observe any statistical difference in 4OI on animal deaths in control mice during endotoxemia or CLP-induced sepsis (Figures 6A and 7A). However, the protection against septic death experienced by Sting1 MyeÀ/À mice was reversed by the administration of itaconate ( Figures 6A  and 7A). Accordingly, plasma HMGB1, SQSTM1, ALT, and BUN in septic Sting1 MyeÀ/À mice were elevated following 4OI treatment ( Figures 6C-6F and 7C-7F). In vitro study further revealed that 4OI at a superphysiologic level directly induced cell death and the release of DAMPs (HMGB1 and SQSTM1) in peritoneal macrophages ( Figures 6I-6K). Overall, these studies indicate that STING1-mediated itaconate production promotes, rather than inhibits, the development of sepsis.

DISCUSSION
TLRs are evolutionally conserved pattern recognition receptors that detect specific PAMPs to active innate immune responses (Akira and Takeda, 2004). In this study, we found a regulatory mechanism for ACOD1 expression by coupling TLR and STING1 signals ( Figure 7I). The activation of STING1 alone by ligands or a gain-of-function mutation is not sufficient to trigger ACOD1 expression. However, activated STING1 leads to increased sensitivity and response to inducible ACOD1 expression and itaconate production following stimulation with several TLR ligands, including LPS. Our findings not only provide insights into the regulation mechanism of immunometabolism (Jung et al., 2019;O'Neill et al., 2016), but also challenge current views on the anti-inflammatory activity of itaconate in vivo (Liao et al., 2019;Mills et al., 2018).
Although ACOD1 was originally described as an LPS-inducing gene in 1995 (Lee et al., 1995), the key function of ACOD1 in mediating itaconate production has not been studied until recently . ACOD1 is a mitochondrial protein, mainly expressed in myeloid cells, and its inducible expression can be used as a marker and regulator of inflammation during various infections . We proved that the STING1-MYD88 complex is a signal hub that mediates inducible ACOD1 expression in response to ligands of TLR1, TRL2, TRL4, TRL5, or TLR6. The function of STING1 in mediating LPS-induced ACOD1 expression depends on MYD88, rather than CGAS, which indicates that the STING1 signal pathway contributes to gene expression under different stimulations.
Signal transduction is a complex process that depends on stimuli and environment. Although CGAS was originally found to be required for STING1 activation during viral infection, recent studies have also reported a CGAS-independent STING1 pathway in response to different stimuli, including viral infection (Holm et al., 2016;Suschak et al., 2016;Unterholzner and Dunphy, 2019). Our current study suggests that MYD88 plays an alternative role in mediating STING1 activity in response to TLR ligands. cGAMP or other ligands of STING may also be produced in a CGAS-independent manner (Carozza et al., 2020;Wan et al., 2020). In fact, Sting À/À and Cgas À/À mice have overlapping and distinct phenotypes in disease models of infection and immunity (Suschak et al., 2016;Yum et al., 2021). An increasing number of natural or synthetic STING1 ligands have been discovered. It is still not excluded that CGAS may be involved in STING1-dependent ACOD1 expression under certain conditions, especially in pathological DNA damage situations (Motwani et al., 2019).
We provide experimental evidence that STING1 forms a protein complex with MYD88, which is necessary for the inducible expression of ACOD1. These findings may also establish a model to explain the interaction of STING1 and TLR signaling in the production of pro-inflammatory cytokines during infection (Tesser et al., 2021;Zeng et al., 2017). Although previous studies have shown that ACOD1 is implicated in the antiviral response (Daniels et al., 2019;Ren et al., 2016), we did not observe that TLR3, TLR7, TLR8, and TLR9 ligands induce ACOD1 expression in human monocytes. Thus, the production of ACOD1 in virus infection may not come directly from nucleic acid ligands. In contrast, viral infection-related cytokines, such as IFNs, likely are a stimulator of ACOD1 upregulation (Degrandi et al., 2009). Regardless, the STING1-MYD88 complex plays a major role in mediating ACOD1 expression during bacterial infection, especially in response to TLR1/2/4/5/6 signals. The degradation of STING1 protein is a posttranslational modification mechanism that can inhibit an excessive innate immunity response (Pokatayev et al., 2020;Prabakaran et al., 2018). Our current study highlights the mechanism by which the formation of the STING1-MYD88 complex prevents STING1 degradation through an autophagic pathway, instead of the ubiquitin-proteasome system. Consequently, inhibiting the autophagic degradation of STING1 increases the level of STING1, providing a priming signal for subsequent MYD88-mediated ACOD1 expression. As STING1 also promotes autophagy (Gui et al., 2019;Zhang et al., 2021), it may establish negative feedback to control the expression and activation of STING1 during infection (Zhang et al., 2022b).
Using the 3 0 3'-cGAMP/LPS stimulation model, we further investigated the downstream transcription factors responsible for ACOD1 expression. Our data suggest that the transcription factor IRF3 coupled with JUN contributes to 3 0 3 0 -cGAMP/LPS-induced ACOD1 expression. In contrast, NF-kB (a well-known pro-inflammatory transcription factor in TLR signaling) is not required for this process. It is important to further identify the nuclear cofactors that facilitate the activation of IRF3 and JUN in controlling 3 0 3 0 -cGAMP/LPS-induced ACOD1 expression. Under different immune signal stimulation, the expression of inducible ACOD1 may depend on different transcription factors .
Our animal study raised a concern about the application of 4OI in infectious diseases. An initial study showed that 4OI has a mild protective effect on LPS-induced lethality in mice (Mills et al., 2018). The anti-inflammatory activity of itaconate or 4OI involves multiple mechanisms, such as the blocking of succinate dehydrogenase activity to reduce succinate-mediated inflammatory processes (Lampropoulou et al., 2016), upregulation of activating transcription factor 3 (ATF3) expression to limit IkBz-mediated pro-inflammatory cytokine production (Bambouskova et al., 2018), or increasing nuclear factor erythroid 2-like 2 (NFE2L2) protein stability to induce anti-inflammatory gene expression (Mills et al., 2018). However, our sepsis mouse model did not find the protective activity of 4OI in a lethal infection. One possible explanation for this discrepancy could be owing to different infection models and timing of 4OI administration. Of note, 4OI treatment reversed the protection against septic death experienced by Sting1 MyeÀ/À mice. Although the mechanism is not clear, we demonstrated that itaconate causes cell death and DAMP (HMGB1 and SQSTM1) release, which is consistent with recent studies on the cytotoxicity of itaconate on cancer cells (Belosludtsev et al., 2020;Qu et al., 2021). HMGB1 and SQSTM1 are potential therapeutic targets for infection as well as sterile inflammation caused by tissue damage (Kang et al., 2014;Wang et al., 1999;Zhou et al., 2020;Zou et al., 2020).
In summary, the activation of the STING1 pathway in monocytes and macrophages can synergize with the MYD88 pathway to drive LPS-induced ACOD1 expression and itaconate production, which favors the development of septic death by the release of DAMPs. This innate immunity pathway may enhance our understanding of the immunopathological mechanisms of lethal infections.

Limitations of the study
A limitation of our work is the use of cell lines rather than primary cells to study the relationship between MYD88 and STING1 in innate immunity. We also cannot rule out whether the MYD88-STING1 pathway is required for ACOD1-related inflammatory responses in other infectious diseases or tissue damage.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Lead contact
Further information should be directed to and will be fulfilled by the lead contact Dr. Daolin Tang (daolin. tang@utsouthwestern.edu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
Data: The authors declare that all data supporting the findings of this study are available within the article or available from the corresponding author upon reasonable request.
Code: This study did not generate any code.
Other items: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell culture